POLQ inhibitor and application thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to a POLQ inhibitor and application thereof. According to the invention, a fluorescence quenching high-throughput screening system is constructed, and a POLQ inhibitor, namely tannic acid (TA), for inhibiting the activity of POLQ polymerase is screened from 3893 compounds. Experiments prove that tannic acid can specifically inhibit the activity of POLQ polymerase, prevent extension of DNA polymerase and inhibit the activity of an MMEJ repair pathway, so that the DNA damage level of homologous recombination repair defective cells is increased, cell proliferation is inhibited, and the effect of resisting cancer cell growth is achieved. The invention provides a novel scheme with high efficiency, low toxicity and difficult drug resistance for targeted therapy of homologous recombination repair of defective tumors, and has important application value in the aspect of development of antitumor drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to a POLQ inhibitor and application thereof. BACKGROUND

[0002] In the process of cell life activities, in order to maintain the stability of the genome, cells have evolved a variety of DNA damage repair (DDR) pathways. DNA double-strand break damage is the most serious type of DNA damage in cells, and its repair pathways mainly include homologous recombination (HR), non-homologous end joining (NHEJ), and microhomology-mediated end-joining (MMEJ). In recent years, people have a deeper understanding of this complex regulatory system of DDR pathway, and more and more DDR-targeted antitumor drugs have been developed for multiple members of the DDR pathway, such as PARP, ATM, ATR, CHK1, WEE1 and DNA-PK, some of which have entered the clinical trial stage. For example, PARP (poly(ADP-ribose) polymerase) inhibitors have a breakthrough therapeutic effect in BRCA gene-mutated tumors, and PARP has become a target for synthetic lethality therapy in homologous recombination-deficient tumors. PARP inhibitor Olaparib was approved by FDA in 2014 as a treatment drug for BRCA gene-mutated tumors, becoming the first successful marketed drug based on synthetic lethality.

[0003] Studies have found that DNA polymerase theta (POLtheta or POLQ) is another important target for synthetic lethality in homologous recombination repair (HR) defects. When HR function is defective, cells will rely more on MMEJ to repair DNA damage, at which time the role of POLQ is particularly critical. Inhibition of POLQ activity will block the MMEJ repair pathway, leading to ineffective repair of DNA damage in HR-deficient cells, increased DNA damage levels, and inhibition of cell proliferation. Therefore, POLQ inhibitors are expected to become an effective and safe new treatment strategy. In addition, POLQ inhibitors can also improve the sensitivity of tumor cells to radiotherapy, improve the response to radiotherapy in animal tumor models, and improve survival rate. In summary, POLQ has become an important target for the treatment of some diseases. The development of compounds that can inhibit the polymerase activity of POLQ has an important application prospect for improving the effectiveness of cancer treatment. The existing POLQ polymerase inhibitors target high pocket homogeneity, which is prone to cause drug resistance due to mutation, and the development of POLQ inhibitors targeting new binding pockets has great prospects for drug development. SUMMARY

[0004] The technical problem solved by the embodiments of the present application is to provide the application of tannic acid in the preparation of a drug for preventing and / or treating diseases.

[0005] The technical problem solved by the embodiments of the present application is to provide a drug for treating diseases.

[0006] The technical problem solved by the embodiments of the present application is to provide a POLQ inhibitor, which can specifically act on inhibiting the growth of cancer cells, and has a broad application prospect in the preparation of anti-tumor related drugs.

[0007] To solve the above technical problems, the technical solutions adopted by the present application are as follows:

[0008] In a first aspect, the embodiments of the present application provide the application of tannic acid in the preparation of a drug for preventing and / or treating diseases.

[0009] The diseases include diseases related to the POLQ target point.

[0010] Further, the diseases related to the POLQ target point include tumors.

[0011] The tumors include tumors with defects in DNA double-strand breakage damage repair.

[0012] In some embodiments, the tumors include tumors with defects in homologous recombination repair.

[0013] In some embodiments, the tumors include tumors with BRCA gene mutations.

[0014] In some embodiments, the tumors with BRCA gene mutations include tumors with BRCA1 and / or BRCA2 gene mutations.

[0015] In some embodiments, the gene mutations include at least one of base substitution mutations, frame shift mutations, deletion mutations, and insertion mutations.

[0016] In some embodiments, the tumors include cancers.

[0017] In some embodiments, the cancers include at least one of colorectal adenocarcinoma, breast cancer, cervical cancer, ovarian cancer, prostate cancer, and pancreatic cancer.

[0018] In a second aspect, the embodiments of the present application further provide a drug for treating diseases, wherein the active ingredient of the drug includes tannic acid (TA).

[0019] In some embodiments, the disease comprises a disease associated with a POLQ target.

[0020] In some embodiments, the disease associated with a POLQ target comprises a tumor.

[0021] In some embodiments, the tumor comprises a tumor with a defect in DNA double-strand break damage repair.

[0022] In some embodiments, the tumor comprises a tumor with a defect in homologous recombination repair.

[0023] In some embodiments, the tumor comprises a tumor with a BRCA gene mutation.

[0024] In some embodiments, the tumor with a BRCA gene mutation comprises a tumor with a BRCA1 and / or BRCA2 gene mutation.

[0025] In some embodiments, the gene mutation comprises at least one of a base substitution mutation, a frameshift mutation, a deletion mutation, an insertion mutation.

[0026] In some embodiments, the tumor comprises a cancer.

[0027] In some embodiments, the cancer comprises at least one of colorectal adenocarcinoma, breast cancer, cervical cancer, ovarian cancer, prostate cancer, pancreatic cancer.

[0028] In some embodiments, the drug is a drug that specifically inhibits DNA polymerase theta activity in the disease, thereby inhibiting tumor cell proliferation.

[0029] In some embodiments, the drug is a DNA polymerase theta inhibitor.

[0030] Further, the embodiments of the present application also provide a drug for treating a disease, wherein an active ingredient of the drug is tannic acid.

[0031] In some embodiments, the disease comprises a disease associated with a POLQ target.

[0032] In some embodiments, the disease associated with a POLQ target comprises a tumor.

[0033] In some embodiments, the tumor comprises a tumor with a defect in DNA double-strand break damage repair.

[0034] In some embodiments, the tumor comprises a tumor with a defect in homologous recombination repair.

[0035] In some embodiments, the tumor comprises a tumor with a BRCA gene mutation.

[0036] In some embodiments, the tumor with BRCA gene mutation comprises a tumor with BRCA1 and / or BRCA2 gene mutation.

[0037] In some embodiments, the gene mutation comprises at least one of base substitution mutation, frame shift mutation, deletion mutation, and insertion mutation.

[0038] In some embodiments, the tumor comprises a cancer.

[0039] In some embodiments, the cancer comprises at least one of colorectal adenocarcinoma, breast cancer, cervical cancer, ovarian cancer, prostate cancer, and pancreatic cancer.

[0040] In some embodiments, the drug is a DNA polymerase theta inhibitor.

[0041] In some embodiments, the drug is a DNA polymerase theta inhibitor.

[0042] In some embodiments, the DNA polymerase theta is POLQ.

[0043] In a third aspect, an embodiment of the present application provides a POLQ inhibitor.

[0044] In some embodiments, the POLQ inhibitor is a substance capable of inhibiting the activity of POLQ.

[0045] In some embodiments, the POLQ inhibitor is capable of inhibiting the polymerase activity of the POLQ protein.

[0046] In some embodiments, the POLQ inhibitor is tannic acid.

[0047] In some embodiments, the tannic acid specifically inhibits the POLQ polymerase and has no inhibitory effect on Taq DNA polymerase and Bsu DNA polymerase. This property makes tannic acid highly specific in inhibiting POLQ polymerase, reduces the interference with the functions of other normal DNA polymerases, and reduces the risk of drug side effects.

[0048] Further, an embodiment of the present application also provides the use of the tannic acid in the preparation of a preparation for inhibiting the proliferation of tumor cells with homologous recombination repair deficiency.

[0049] In some embodiments, the tannic acid inhibits the activity of the MMEJ repair pathway by inhibiting the activity of the POLQ polymerase, thereby effectively inhibiting the proliferation of tumor cells with homologous recombination repair deficiency.

[0050] In some embodiments, the tumor cells with homologous recombination repair deficiency comprise tumor cells with BRCA1 and / or BRCA2 gene mutation.

[0051] In some embodiments, the homologous recombination repair deficient tumor cells comprise tumor cells with BRCA1 and / or BRCA2 gene deletion.

[0052] In some embodiments, the homologous recombination repair deficient tumor cells are derived from at least one of colorectal adenocarcinoma cells, breast cancer cells, cervical cancer cells, ovarian cancer cells, prostate cancer cells, and pancreatic cancer cells.

[0053] In some embodiments of the present application, the homologous recombination repair deficient tumor cells comprise colorectal adenocarcinoma cells with BRCA2 gene deletion: DLD1 BRCA2- / - cells.

[0054] Further, the embodiments of the present application also provide a preparation for inhibiting proliferation of homologous recombination repair deficient tumor cells.

[0055] In some embodiments, the effective active ingredient in the preparation comprises tannic acid.

[0056] Preferably, the effective active ingredient in the preparation is tannic acid.

[0057] In some embodiments, the homologous recombination repair deficient tumor cells comprise tumor cells with BRCA1 and / or BRCA2 gene mutation.

[0058] In some embodiments, the homologous recombination repair deficient tumor cells comprise tumor cells with BRCA1 and / or BRCA2 gene deletion.

[0059] In some embodiments, the homologous recombination repair deficient tumor cells comprise at least one of colorectal adenocarcinoma cells, breast cancer cells, cervical cancer cells, ovarian cancer cells, prostate cancer cells, and pancreatic cancer cells.

[0060] In some embodiments of the present application, the homologous recombination repair deficient tumor cells comprise colorectal adenocarcinoma cells with BRCA2 gene deletion: DLD1 BRCA2- / - cells.

[0061] In some embodiments, the preparation is in any dosage form acceptable in the pharmaceutical field, including but not limited to any of powder, granules, injection, capsule, tablet, oral liquid.

[0062] In some embodiments, the preparation is administered in any of oral, intravenous injection, intramuscular injection.

[0063] The preparation further comprises pharmaceutically acceptable adjuvants, including but not limited to surfactants, suspending agents, emulsifying agents and some new pharmaceutical polymer materials, such as cyclodextrin, chitosan, polylactic acid (PLA), polylactic acid glycolic acid copolymer (PLGA), hyaluronic acid and the like, which are used to improve the stability, solubility, release characteristics and the like of the drug.

[0064] Further, the embodiment of the present application also provides a high-throughput screening method of the POLQ inhibitor, comprising the following steps:

[0065] (i) annealing and pairing DNA;

[0066] (ii) mixing and incubating the compound to be screened with the POLQ protein, and then adding the reaction solution containing the template DNA to start the reaction;

[0067] (iii) adding EDTA to the incubated reaction solution to terminate the polymerase extension reaction;

[0068] (iv) screening the preliminary screening compound by detecting the FAM fluorescence signal;

[0069] (v) further reducing the final concentration of the compound to be screened, and repeating the above screening process for high-throughput screening.

[0070] The reaction solution comprises MgCl2 and dNTP.

[0071] In some embodiments of the present application, the high-throughput screening method of the POLQ inhibitor specifically comprises the following steps:

[0072] (I) heating the DNA mixture solution at 95℃ for 5 minutes, and then slowly cooling to 25℃ to complete annealing and pairing;

[0073] (II) incubating the compound to be screened at a final concentration of 30 μM with 20 nM POLQ protein at room temperature for 30 minutes, and then adding an equal volume of the DNA mixture solution of step (I), wherein the reaction solution comprises MgCl2 at a final concentration of 10 mM and dNTP at a final concentration of 50 μM, and reacting at 37℃ for 60 minutes;

[0074] (III) adding EDTA at a final concentration of 25 mM to step (II) to terminate the reaction;

[0075] (IV) detecting the FAM fluorescence signal by using an enzyme label instrument, calculating the fluorescence signal value and the inhibition rate, taking the inhibition rate ≥ 50% as the screening standard, and obtaining the preliminary screening compound;

[0076] (V) reducing the final concentration of the compound to be screened to 10 μM, repeating the processes of steps (II)-(IV), and screening the preliminary screening compound to obtain the re-screening compound.

[0077] (VI) reducing the final concentration of the compound to be screened to 2 μM, repeating the process of steps (II)-(IV) to re-screen the re-screened compound, and obtaining a candidate inhibitor molecule;

[0078] (VII) determining the IC50 of the candidate inhibitor molecule obtained in step (VI) for inhibiting the POLQ polymerase, and obtaining the POLQ inhibitor of claim 1. 50 concentration and specificity, and screening obtains the POLQ inhibitor of claim 1.

[0079] The high-throughput screening method has a fluorescence quenching high-throughput screening system with a signal-to-noise ratio of 11, which is better than a traditional PicoGreen dye method (a signal-to-noise ratio of 3.5), and the sensitivity is significantly improved by more than 3 times.

[0080] In step (I), the DNA mixture is obtained by dissolving primers with a final concentration of 0.11 μM, FAM-labeled template DNA with a final concentration of 0.1 μM, and BHQ1-labeled primers with a final concentration of 0.11 μM in a buffer.

[0081] Specifically, the nucleotide sequence of the primers is shown in SEQ ID NO. 3; the nucleotide sequence of the FAM-labeled template DNA is shown in FAM+SEQ ID NO. 5; and the nucleotide sequence of the BHQ1-labeled primers is shown in BHQ1+SEQ ID NO. 5.

[0082] In step (II), the POLQ protein is a purified POLQ-pol protein.

[0083] Specifically, the POLQ-pol protein is obtained by optimizing the codons preferred by Escherichia coli for the sequence of the polymerase domain protein of the POLQ gene, then using seamless cloning to clone the synthesized target fragment into a vector pET-28a constructed in the experiment, which has a polyhistidine and SUMO fusion tag at the N terminus and a StrepII tag at the C terminus, to obtain a recombinant protein plasmid pET-28a-6XHis-SUMO-POLQ-pol-Strep II, and then transforming the recombinant protein plasmid into an Escherichia coli expression Rosetta (DE3) strain to obtain the protein after induction and expression.

[0084] Specifically, the POLQ-pol protein has DNA polymerase activity.

[0085] In step (II), the compound to be screened is, in some embodiments of the present application, 3,893 compounds in a drug compound library and a clinical compound library.

[0086] In step (IV), the FAM fluorescence signal is detected by an enzyme label instrument, specifically, a multifunctional enzyme label instrument is used to detect the FAM fluorescence signal value at 420 nm excitation and 520 nm emission.

[0087] Further, through the in vitro biochemical MMEJ report system and the U2OS MMEJ report cell line, it is found that the preferred POLQ inhibitor, tannic acid, can inhibit the activity of the MMEJ repair pathway in vitro and in cells.

[0088] Further, it is found that the preferred POLQ inhibitor of the application, tannic acid, can inhibit the proliferation of BRCA2 knockout cells, inhibit the increase of DNA damage signals in BRCA2 knockout cells caused by POLQ, and inhibit the clonogenicity of BRCA2 knockout DLD1 tumor cells, indicating that tannic acid has a significant inhibitory effect on cell proliferation on BRCA2-deficient tumor cells by inhibiting the activity of POLQ polymerase.

[0089] In the following examples, the cells used to explore the tannic acid inhibition of BRCA2 knockout cell proliferation include but are not limited to the RPE-1 BRCA2- / -#1 cells, RPE-1 BRCA2- / -#2 cells, and DLD1 BRCA2 knockout cells (DLD1 BRCA2- / - cells) independently constructed by the laboratory. As long as the cells can be used to explore the tannic acid inhibition of BRCA2 knockout cell proliferation in the prior art, they can be used in some embodiments of the application.

[0090] Beneficial effects:

[0091] (1) The embodiments of the application obtain the POLQ inhibitor, tannic acid, which specifically inhibits the activity of POLQ polymerase, by constructing a fluorescence quenching high-throughput and high signal-to-noise ratio screening system from 3893 compounds.

[0092] (2) The embodiments of the application find a new binding pocket of tannic acid and POLQ polymerase, which avoids the current problem of high homogeneity of the reported inhibitor binding pocket and the drug resistance caused by pocket mutation, and improves the therapeutic effect and application range of the drug.

[0093] (3) The POLQ inhibitor of the embodiments of the application can specifically inhibit the activity of POLQ polymerase, prevent the extension of DNA polymerase, inhibit the activity of the MMEJ pathway, increase the DNA damage level of homologous recombination repair-deficient tumor cells, inhibit the proliferation of BRCA2-deficient cells, and thus has an anticancer cell growth effect, which has a broad application prospect in the development of antitumor drugs.

[0094] (4) The embodiment of the present application provides a new scheme of high efficiency, low toxicity and non-drug resistance for targeted treatment of homologous recombination repair deficiency (HRD) tumors, and uses tannic acid or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof as an active ingredient, can selectively block the MMEJ (microhomology-mediated end joining) repair pathway, induce synthetic lethal effect of homologous recombination repair deficiency tumor cells, and provide strong support for targeted treatment of HRD tumors. BRIEF DESCRIPTION OF DRAWINGS

[0095] The above and / or other aspects of the present application will become apparent from the following more detailed description of the application, taken in conjunction with the accompanying drawings.

[0096] Figure 1 POLQ-pol protein purification and polymerase activity verification. Wherein A is the purified POLQ-pol DNA polymerase protein domain; B is the primer extension experiment for detecting the polymerase activity mode diagram; C is the POLQ-pol DNA polymerase activity detection; D is that ART558 has a specific inhibitory effect on the POLQ-pol DNA polymerase activity; E is the primer extension property of POLQ-pol DNA polymerase.

[0097] Figure 2 The fluorescence quenching screening system and the PicoGreen method are compared in terms of signal-to-noise ratio. Wherein A is the principle of PicoGreen dye method for detecting POLQ-pol polymerase activity; B is the signal-to-noise ratio of PicoGreen dye method for detecting POLQ-pol polymerase activity; C is the principle of fluorescence quenching system for detecting POLQ-pol polymerase activity; D is the signal-to-noise ratio of fluorescence quenching system for detecting POLQ-pol polymerase activity.

[0098] Figure 3 High-throughput screening of the inhibition rate of candidate molecules on POLQ-pol polymerase activity. Wherein A is the use of marketed drug compound library and clinical phase molecular compound library to screen POLQ-pol DNA polymerase inhibitors (compound concentration 30 μM); B is the inhibitory effect of 10 μM concentration of candidate compounds on POLQ-pol DNA polymerase activity; C is the inhibitory effect of 2 μM concentration of candidate compounds on POLQ-pol DNA polymerase activity.

[0099] Figure 4 Inhibition activity IC of tannic acid on POLQ-pol 50 determination and specificity verification. Wherein A is the detection result of the influence of tannic acid on POLQ-pol DNA polymerase activity; B is the IC 50Fitting calculation results; C is the detection result of the effect of tannic acid on Taq DNA polymerase activity; D is the detection result of the effect of tannic acid on Bsu DNA polymerase activity.

[0100] Figure 5 Tannic acid and POLQ binding pocket prediction. Among them, A is ART558 and POLQ-pol binding pocket; B is RTX-152 and POLQ-pol binding pocket; C is the prediction result of tannic acid and POLQ-pol binding pocket.

[0101] Figure 6 Tannic acid inhibits POLQ-pol protein MMEJ activity in vitro. Among them, A is the detection mode diagram of POLQ-pol in vitro mediated MMEJ activity; B is the tannic acid in vitro inhibition of POLQ-pol mediated MMEJ activity.

[0102] Figure 7 Tannic acid inhibits MMEJ activity in cells.

[0103] Figure 8 Tannic acid inhibits BRCA2 knockout cell proliferation.

[0104] Figure 9 Tannic acid inhibits POLQ to increase DNA damage level in BRCA2 knockout cells.

[0105] Figure 10 Tannic acid inhibits BRCA2 knockout tumor cell colony formation. DETAILED DESCRIPTION

[0106] The above and / or other aspects of the present application will become apparent and more readily appreciated from the following description, taken in conjunction with the accompanying drawings, in which:

[0107] The experimental methods described in the following examples are all routine methods unless otherwise specified; the reagents and materials described in the following examples are all commercially available unless otherwise specified.

[0108] Example 1: POLQ-pol protein purification and polymerase activity verification

[0109] I. POLQ-pol gene cloning and protein purification

[0110] The target gene sequence of POLQ-pol protein was obtained from the NCBI website, and the polymerase domain protein sequence of POLQ-pol gene was optimized for E. coli preferred codons. The optimized sequence was entrusted to Jiutian Gene Company for synthesis. The optimized nucleotide sequence is shown in SEQ ID NO. 1, and the amino acid sequence is shown in SEQ ID NO. 2. The synthesized target fragment was cloned into the vector pET-28a with a polyhistidine and SUMO fusion tag at the N terminus and a StrepII tag at the C terminus constructed in the laboratory by seamless cloning. After sequencing, the correct cloning was identified, and the recombinant protein plasmid pET-28a-6xHis-SUMO-POLQ-pol-Strep II was obtained. The nucleotide sequence is shown in SEQ ID NO. 3.

[0111] The recombinant protein plasmid pET-28a-6XHis-SUMO-POLQ-pol-Strep II was transformed into E. coli expression strain Rosetta (DE3), ice bath for 20 minutes, 42℃ heat shock for 45 seconds, ice bath for 2 minutes, then add 500 μL LB liquid recovery for 1 hour, then spread the recovered bacterial liquid on agarose plates containing 50 μg / mL kanamycin and incubate at 37℃ overnight. The positive colonies were picked and cultured in fresh LB medium containing kanamycin. The expression strain was activated overnight, then transferred to fresh LB liquid medium containing kanamycin at a ratio of 1% v / v, and incubated at 220 rpm and 37℃ for about 2-3 hours. When the concentration of the bacterial liquid was about 0.4-0.6, the final concentration of inducer IPTG was added to 0.5 mM, and the culture was induced at 16℃ overnight. After incubation, the bacterial cells were collected by centrifugation, resuspended with lysis buffer (formula: 50 mM Tris-HCl pH 7.4, 300 mM NaCl, 10% glycerol) and added with PMSF at a final concentration of 1 mM. The bacteria were broken by low-temperature high-pressure homogenizer, and the broken solution was clarified by centrifugation at 15000g for 30 minutes at 4℃. The supernatant was filtered through a 0.4 μm filter, purified by Strep II tag affinity chromatography, and the target protein was eluted by desulfitobiotin competition. The specific method is described in the affinity column instruction. After purification of the target protein POLQ-pol, it was mixed with 5x SDS protein loading buffer (formula: 50 mM Tris-HCl pH 6.8, 10% (w / v) SDS, 50% (v / v) glycerol, 5% (v / v) β-mercaptoethanol) at 95℃ for 10 minutes, and the purification effect of the target protein POLQ-pol was detected by SDS-PAGE. The purification result is shown in Fig. A of Figure 1

[0112] II. Verification of POLQ-pol protein polymerase activity​

[0113] Polymerase primer extension assay was applied to detect the DNA polymerase activity of purified POLQ-pol protein by polymerase dependent DNA template primer extension to synthesize full-length extension product (schematic diagram as shown in FIG. 1B). The specific procedure is as follows: Figure 1

[0114] 1. Primer / template annealing

[0115] The final concentration of 0.11 μM FAM-labeled primer (FAM+SEQ ID NO. 4, wherein SEQ ID NO. 4: 5'-CCAGCCTGCGGCGAGTG-3') and the final concentration of 0.1 μM template DNA (SEQ ID NO. 5: 5'-ACTCTTCTTCTCTTCTTCACTCGCCGCAGGCTGG-3') were dissolved in buffer (50 mM Tris-HCl buffer (pH 7.4), 50 mM NaCl), and then the DNA mixture was heated at 95 °C for 5 minutes and slowly cooled to 25 °C to complete the annealing pairing.

[0116] 2. Polymerase extension reaction

[0117] The final concentration of 10 mM MgCl2, the final concentration of 50 μM dNTP Thermo Scientific, and the final concentration of 0.15 U / μL Bsu DNA Polymerase (NEB) or Taq DNA polymerase (Thermo) or the final concentration of 50 nM, 100 nM purified POLQ-pol protein were added, and then the DNA polymerase extension was performed at 37 °C for 60 minutes, and the reaction was terminated with the final concentration of 25 mM EDTA.

[0118] 3. Product purification and denaturation

[0119] The sample after step 2 reaction was digested with proteinase K at 37 °C for 30 minutes, and an equal volume of extraction solution (the volume ratio of phenol: chloroform: isoamyl alcohol = 25:24:1) was added for extraction, and then centrifuged at 12000 rpm for 10 minutes, and 10 μL of DNA solution was taken from the supernatant and added to 30 μL of deionized formamide, and then heated at 95 °C for 5 minutes for denaturation.

[0120] 4. Gel electrophoresis and imaging

[0121] ​Prepare denatured polyacrylamide gel with 8M urea at a concentration of 12%, take 10 μL of the heated denatured DNA sample from step 3 and load it into the gel, use 1X TBE as the electrophoresis buffer, and run the electrophoresis at 30V / cm for 20 min, then use the Bio-Rad gel imager to image.

[0122] The detection results are shown in Figure 1 As shown in FIG. C, compared with Taq DNA polymerase (lane 3) and Bsu DNA polymerase (lane 4), the addition of 50 nM and 100 nM POLQ-pol protein can detect full-length DNA extension products (lanes 5 and 6), which indicates that the purified POLQ-pol protein has DNA polymerase activity.

[0123] In order to further confirm that the observed polymerase activity is caused by the purified POLQ-pol protein and not by the residual polymerase activity of other impurity proteins, the reported POLQ polymerase activity inhibitor ART558 was added to the polymerase extension system at 0.1 μM, 1 μM and 10 μM, and compared with the POLQ-pol protein without the addition of the inhibitor. After the addition of ART558, the full-length extension product of POLQ-pol was completely inhibited with the increase of the concentration of ART558 inhibitor, while ART558 had no inhibitory effect on Bsu DNA polymerase (FIG. D in Figure 1 In addition, the effect of the reaction time of the polymerase extension on the polymerase activity of POLQ-pol protein was further studied, and it was found that the polymerase activity of POLQ-pol protein increased with the increase of the reaction time, and the proportion of the full-length extension product increased, and the proportion of the full-length extension product basically reached saturation after about 10 minutes (FIG. E in Figure 1

[0124] Example 2: Comparison of signal-to-noise ratio between fluorescence quenching screening system and PicoGreen method

[0125] The above polymerase primer extension detection results confirm that the purified POLQ-pol protein has DNA polymerase activity and can be specifically inhibited by the commercial inhibitor ART558, but this method is based on the detection of DNA gel electrophoresis of the extension product, which is not suitable for high-throughput POLQ-pol inhibitor screening work. Therefore, the embodiment of the present application establishes a set of fluorescence quenching system, which utilizes the strand displacement activity of the DNA chain extended by POLQ-pol polymerase. When strand displacement occurs during the extension of POLQ-pol polymerase, the BHQ1 quenching group no longer quenches the FAM group on the DNA template strand, and then the activity of DNA polymerase is reflected by the strength of the FAM group fluorescence signal (as shown in FIG. B in Figure 2

[0126] The specific fluorescence quenching detection process is as follows:​​

[0127] 1. Dissolve 0.11 μΜ of primer (SEQ ID NO. 4: 5'-CCAGCCTGCGGCGAGTG-3'), 0.1 μΜ of FAM-labeled template DNA (FAM + SEQ ID NO. 6, wherein SEQ ID NO. 6: 5'-CACTGTGAGCTTAGCTCACATTTCACTCGCCGCAGGCTGG-3') and 0.11 μΜ of BHQ1-labeled primer (BHQ1 + SEQ ID NO. 7, wherein SEQ ID NO. 7: 5'-CTAAGCTCACA GTG-3') in buffer (50 mM Tris-HCl (pH 7.4), 50 mM NaCl), then heat the DNA mixture at 95 °C for 5 minutes, slowly cool to 25 °C, and complete annealing pairing.

[0128] 2. Add 10 mM of MgCl2, 50 μΜ of dNTP (Thermo Scientific) and 20 nM of purified POLQ-pol protein, then perform DNA polymerase extension at 37 °C for 60 minutes, and terminate the reaction with 25 mM of EDTA.

[0129] 3. Detect FAM fluorescence signal value at 420 nm excitation and 520 nm emission using a multifunctional enzyme marker, and calculate fluorescence signal change by GraphPad.

[0130] As a control, PicoGreen dye method was used, and the specific detection process was as follows:

[0131] (1) Dissolve 0.11 μΜ of primer (SEQ ID NO. 4: 5'-CCAGCCTGCGGCGAGTG-3') and 0.1 μΜ of template DNA (SEQ ID NO. 6: 5'-CACTGTGAGCTTAGCTCACATTTCACTCGCCGCAGGCTGG-3') in buffer (50 mM Tris-HCl buffer (pH 7.4), 50 mM NaCl), then heat the DNA mixture at 95 °C for 5 minutes, slowly cool to 25 °C, and complete annealing pairing.

[0132] (2) Add 10 mM of MgCl2, 50 μΜ of dNTP (Thermo Scientific) and 20 nM of purified POLQ-pol protein, then perform DNA polymerase extension at 37 °C for 60 minutes, and terminate the reaction with 25 mM of EDTA.

[0133] (3) Add PicoGreen dye (Thermo) to the reaction system at a final concentration of 1* to detect double-stranded DNA extension products.

[0134] (4) Use a multifunctional enzyme marker to detect the Picogreen fluorescence signal value at 420 nm excitation and 520 nm emission, and use GraphPad to plot and calculate the fluorescence change.

[0135] The detection results are shown in Figure 2 Compared with the traditional PicoGreen dye method for detecting dsDNA products to indirectly detect POLQ-pol activity (A in Figure 2 ), the fluorescence quenching system established in the present application has a better signal-to-noise ratio. The signal value / background noise signal value after adding POLQ-pol reaction in the PicoGreen dye method is about 3.5 (B in Figure 2 ), and the signal-to-noise ratio of the fluorescence quenching detection is about 11 (D in Figure 2 ), indicating that the screening system established in the present application will have higher screening sensitivity, which is helpful for efficient and high-throughput screening of POLQ-pol inhibitors.

[0136] Example 3: High-throughput screening of the inhibition rate of candidate molecules on POLQ-pol polymerase activity

[0137] Based on the fluorescence quenching screening system established in Example 2, the marketed drug compound library and the clinical phase compound library (3893 compounds) were subjected to high-throughput screening work to screen POLQ-pol polymerase inhibitors.

[0138] 1. First round of screening: select 30 μM compound concentration for screening

[0139] Use Echo instrument to add 3893 compounds into independent wells of 384 multi-well plates, and use a dispenser to add 5 μL of purified POLQ-pol protein at a final concentration of 20 nM into each well, and incubate at room temperature for 30 minutes. Then, add an equal volume of DNA mixture (Step 1 in Example 2) that has been annealed into each well, which contains a final concentration of 10 mM MgCl2 and a final concentration of 50 μM dNTP (Thermo Scientific), and perform DNA polymerase extension at 37°C for 60 minutes, and then terminate the reaction with a final concentration of 25 mM EDTA. Finally, use a multifunctional enzyme marker to detect the FAM fluorescence signal value at 420 nm excitation and 520 nm emission, and use GraphPad to plot and calculate the fluorescence change. Take no POLQ-pol protein as a negative control, and take no compound and add POLQ-pol protein as a positive control.

[0140] The screening criteria for hits is an inhibition rate of > 50%. The results are shown in Figure 3 A of Table 1. A total of 218 candidate molecules were obtained from the primary screening.

[0141] 2. Second round of screening: The concentration of the compounds was sequentially reduced to 10 μM, 2 μM

[0142] The concentration of the compounds was reduced to 10 μM, and the high-throughput screening process was repeated to re-screen the 218 compounds obtained in the first round of screening to confirm the compounds that still had inhibitory activity at a lower concentration. Further, the concentration of the compounds was reduced to 2 μM, and the high-throughput screening process was repeated (B and C of Table 1). Finally, the compounds that still had an inhibitory effect on the POLQ-pol polymerase activity at a concentration of 2 μM were confirmed as candidate inhibitor molecules, and were subjected to further identification and confirmation. Figure 3

[0143] Example 4: IC50 of tannic acid for the inhibitory activity of POLQ-pol 50 Determination and specificity verification

[0144] After obtaining the candidate inhibitor molecules through high-throughput screening, the IC50 of the candidate inhibitor molecule, tannic acid, for the inhibition of POLQ-pol polymerase was further determined in this embodiment 50 . The polymerase extension experiment described in Example 1 was used for the determination, and the results showed that, as the concentration of the compound tannic acid increased, the POLQ-pol polymerase extension product was gradually inhibited (A of Table 2). Fitting calculation showed that the IC50 of the compound tannic acid for the inhibition of POLQ-pol polymerase activity was 36.17 nM (B of Table 2). The compound tannic acid had little effect on the activity of the commercial Taq DNA polymerase and Bsu DNA polymerase (C and D of Table 2), which indicated that the inhibition of tannic acid on the POLQ polymerase activity had good specificity. 50 Figure 4 Figure 4 Figure 4

[0145] Example 5: Prediction of the binding pocket of tannic acid to POLQ-pol

[0146] The above embodiment results confirmed that tannic acid had nM-level in vitro inhibitory activity on POLQ-pol polymerase. The CB-Dock2 software was used to simulate the molecular docking of tannic acid and POLQ-pol. The results are shown in Table 3. Unlike the binding pockets of the reported POLQ-pol inhibitors ART558 and RTX-152 (A and B of Table 3), the optimal binding pocket of tannic acid and POLQ-pol polymerase obtained through simulation docking was pocket 4 (C of Table 3). Figure 5 Figure 5 ​​​​​​​Figure 5 C) in FIG. 6. It is speculated that this is related to the nature of tannic acid itself, the molecular weight and spatial structure of tannic acid are quite different from ART558 and RTX-152, resulting in different binding pockets, and thus the mechanism of tannic acid inhibition of POLQ-pol is speculated to be different from the allosteric effect inhibition mechanism reported in the literature.

[0147] Example 6: Exploration of tannic acid inhibition of MMEJ activity

[0148] 1. Tannic acid inhibits MMEJ activity of POLQ-pol protein in vitro.

[0149] Using an in vitro biochemical MMEJ reporter system, the detection mode diagram is shown in FIG. 5A. The FAM-labeled fluorescent DNA strand (FAM+SEQ ID NO. 8, wherein SEQ ID NO. 8: 5'- CACTGTGAGCTTAGGG GTTAGCCCGGG-3') is annealed and paired with the non-labeled part of the primer (SEQ ID NO. 7: 5'-CTAAGCTCACA GTG-3'). The end of the SEQ ID NO. 8 DNA strand has a CCGG reverse complementary micro-homologous sequence, which can be complementary paired between each other under the action of POL-pol polymerase, and under the action of POL-pol polymerase activity, primer extension and strand displacement reaction are carried out using dNTP to produce longer MMEJ product double-stranded DNA. The product DNA length can be detected by non-denaturing acrylamide gel electrophoresis, as shown in FIG. 5B. Figure 6 Figure 6 As shown in FIG. 5B, compared with the control group (lane 2), with the increase of the concentration gradient of added tannic acid, the MMEJ double-stranded DNA product is gradually inhibited (lanes 3-14), and lane 15 is a positive control group with 1 μM ART558 inhibitor added. The above experimental results show that tannic acid can inhibit the MMEJ activity of POLQ-pol protein in vitro.

[0150] 2. Tannic acid inhibits MMEJ activity in cells.

[0151] Using U2OS MMEJ reporter cell lines, DNA double-strand breaks are induced by adding DOX during cell culture, and tannic acid compounds or ART558 molecules are added, with DMSO added as a control. After 48 hours of DOX induction, the activity of MMEJ is detected by flow cytometry to detect the proportion of GFP-positive cells, and the higher the proportion of positive cells, the stronger the MMEJ activity.

[0152] The results are shown in FIG. 6. Figure 7 ​As shown, with the addition of tannic acid, the activity of MMEJ was inhibited, and this inhibition was dependent on the concentration gradient. The positive control molecule ART558 could also significantly inhibit the MMEJ activity in cells. It was shown that tannic acid could also inhibit MMEJ activity in cells, and the inhibition effect was better than ART558 under the same molecular concentration conditions.

[0153] 1. Tannic acid inhibits the proliferation of BRCA2 knockout cells.

[0154] The effect of tannic acid on cell viability was detected by standard CCK8 method. RPE-1 cells (ATCC) or RPE-1 BRCA2- / -#1 clone and RPE-1 BRCA2- / -#2 clone (constructed in our laboratory) were seeded in 96-well plates at 1000 cells per well. After 12 hours of inoculation, fresh medium (F12 / DMEM fresh medium containing 10% FBS and 100 U / mL penicillin, 100 U / mL streptomycin) was replaced, then different concentrations (0, 0.75, 1.5, 3.125, 6.25, 12.5, 25 μM) of tannic acid were added, and after 48 hours of culture, fresh medium containing different concentrations of tannic acid was replaced for continuous treatment for 48 hours. Then 10 μL of CCK8 solution (purchased from Biyun Tian) was added to each well, and incubated at 37°C for 4 hours. Finally, the absorbance was measured at 420 nm using Biotech multifunctional enzyme marker, and the cell viability calculation analysis was performed using GraphPad Prism software.

[0155] Figure 8 The results showed that with the increase of the concentration of the compound tannic acid, the growth of RPE-1 cells, RPE-1 BRCA2- / -#1 cells and RPE-1 BRCA2- / -#2 cells was inhibited, but under the same concentration of tannic acid, the inhibition effect on RPE-1 BRCA2- / -#1 and RPE-1 BRCA2- / -#2 cells was more obvious, which indicated that there was a synthetic lethal effect between the inhibition of POLQ polymerase activity by tannic acid and BRCA2 deficiency.

[0156] 2. Tannic acid inhibits the increase of DNA damage signals in BRCA2 knockout cells caused by POLQ.

[0157] Tannic acid treatment significantly inhibited the proliferation of RPE-1 BRCA2- / -#1 and RPE-1 BRCA2- / -#2 cells, and further detected the effect of tannic acid on DNA damage repair of RPE-1 cells and RPE-1 BRCA2- / -#1 cells. RPE-1 cells or RPE-1 BRCA2- / -#1 cells were inoculated in a 6-well plate, and after overnight adherent culture, different concentrations of tannic acid were added for 48 hours, and DMSO group was used as control, and the level of γH2AX damage signal was detected by immunoblotting experiment. Figure 9 The experimental results show that after adding 10 μM or 20 μM tannic acid treatment, the BRCA2-deficient cell γH2AX protein level is increased, indicating that the degree of DNA damage is higher, while in wild-type RPE-1 cells, no effect on DNA damage is observed after adding tannic acid treatment. This indicates that in BRCA2-deficient cells, tannic acid inhibits POLQ polymerase activity, resulting in an increase in the overall DNA damage level of the cells, which in turn leads to a synthetic lethal effect between POLQ polymerase activity inhibition and BRCA2 deficiency.

[0158] 3. Tannic acid inhibits BRCA2 knockout DLD1 tumor cell colony formation.

[0159] Logarithmic growth period DLD1 wild type, DLD1 BRCA2 knockout cells (constructed in the laboratory), trypsin digestion into single cell suspension, resuspend the cells with complete culture medium and accurately count, inoculation density: DLD1 wild type cells 300 cells / well, DLD1 BRCA2 knockout cells 1000 cells / well (12-well plate), gently shake to mix, avoid local concentration. After the cells adhere, add tannic acid with a final concentration of 2 μM, DMSO as control group, replace the fresh medium containing or not containing tannic acid every three days, continuously culture for 9-12 days (DLD1 wild type cells, 9 days; DLD1 BRCA2 knockout cells, 12 days), form visible colony cell mass under microscope. After colony formation, discard the culture medium, wash with warm PBS for 3 times, add 1 mL 4% paraformaldehyde per well for 15 minutes at room temperature, discard the fixing solution, wash once with PBS, then add 1 mL 0.5% crystal violet staining solution for 20 minutes, discard the staining solution, rinse with PBS until the background is clean, dry and take a photo on the gel imaging instrument, count the number of colonies, and make a statistical graph by GraphPad software to calculate the relative colony formation rate.

[0160] Figure 10 The results show that the addition of 2 μM tannic acid can significantly inhibit the colony formation of DLD1 BRCA2 knockout cells, but has little effect on DLD1 wild type cells, which also indicates that there is a synthetic lethal effect between tannic acid inhibition of POLQ polymerase activity and BRCA2 deficiency.

[0161] In summary, tannic acid has a significant inhibitory effect on cell proliferation of BRCA2-deficient cells by inhibiting the activity of POLQ polymerase, thereby having an anticancer cell growth effect, and has important application value in the development of antitumor drugs.

[0162] The embodiment of the present application provides a kind of POLQ inhibitor and its application idea and method, the method and approach of specifically realizing this technical scheme are many, above-mentioned only is the preferred embodiment of the present application, it should be pointed out, for the ordinary skilled person in the prior art, without departing from the principle of the present application, can also make several improvements and refinements, these improvements and refinements also should be considered as the protection scope of the present application. The components not explicitly described in the embodiment can be implemented using existing technology.

Claims

1. Use of tannic acid for the preparation of a medicament for the prevention and / or treatment of a disease, wherein, The disease includes a disease related to a POLQ target.

2. Use according to claim 1, characterized in that, The disease related to the POLQ target includes a tumor.

3. Use according to claim 2, characterized in that, The tumor includes a tumor with a defect in DNA double-strand break damage repair. Optionally, the tumor includes a tumor with a defect in homologous recombination repair. Optionally, the tumor includes a tumor with a BRCA gene mutation.

4. Use according to claim 2, characterized in that, The tumor includes a cancer.

5. Use according to claim 4, characterized in that, The cancer includes at least one of colorectal adenocarcinoma, breast cancer, cervical cancer, ovarian cancer, prostate cancer, and pancreatic cancer.

6. A medicament for treating a disease, characterized by, The active ingredient of the drug includes tannic acid. Optionally, the disease includes a disease related to a POLQ target. Optionally, the disease related to the POLQ target includes a tumor. Optionally, the tumor includes a tumor with a defect in DNA double-strand break damage repair. Optionally, the tumor includes a tumor with a defect in homologous recombination repair. Optionally, the tumor includes a tumor with a BRCA gene mutation. Optionally, the tumor includes a cancer. Optionally, the cancer includes at least one of colorectal adenocarcinoma, breast cancer, cervical cancer, ovarian cancer, prostate cancer, and pancreatic cancer.

7. The medicament according to claim 6, characterized in that, The drug is a drug that specifically inhibits the activity of DNA polymerase theta in the disease; optionally, the drug is a DNA polymerase theta inhibitor.

8. A medicament for treating a disease, characterized by, The active ingredient of the drug includes tannic acid. Optionally, the disease includes a disease related to a POLQ target. Optionally, the disease related to the POLQ target includes a tumor. Optionally, the tumor includes a tumor with a defect in DNA double-strand break damage repair. Optionally, the tumor includes a tumor with a defect in homologous recombination repair. Optionally, the tumor includes a tumor with a BRCA gene mutation. Optionally, the tumor includes a cancer. Optionally, the cancer includes at least one of colorectal adenocarcinoma, breast cancer, cervical cancer, ovarian cancer, prostate cancer, and pancreatic cancer.

9. The medicament according to claim 8, characterized in that, The drug is a drug that specifically inhibits the activity of DNA polymerase theta in the disease; optionally, the drug is a DNA polymerase theta inhibitor.

10. A POLQ inhibitor, characterized in that, The POLQ inhibitor is tannic acid.

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